Design of a Multisensory Device for Tomato Volatile Compound Detection Based on a Mixed Metal Oxide-Electrochemical Sensor Array and Optical Reader.

Insufficient control of tomato ripening before harvesting and infection by fungal pests produce large economic losses in world tomato production. Aroma is an indicative parameter of the state of maturity and quality of the tomato. This study aimed to design an electronic system (TOMATO-NOSE) consisting of an array of 12 electrochemical sensors, commercial metal oxide semiconductor sensors, an optical camera for a lateral flow reader, and a smartphone application for device control and data storage. The system was used with tomatoes in different states of ripeness and health, as well as tomatoes infected with Botrytis cinerea. The results obtained through principal component analysis of the olfactory pattern of tomatoes and the reader images show that TOMATO-NOSE is a good tool for the farmer to control tomato ripeness before harvesting and for the early detection of Botrytis cinerea.

Cbt modulates Foxo activation by positively regulating insulin signaling in Drosophila embryos.

In late Drosophila embryos, the epidermis exhibits a dorsal hole as a consequence of germ band retraction. It is sealed during dorsal closure (DC), a morphogenetic process in which the two lateral epidermal layers converge towards the dorsal midline and fuse. We previously demonstrated the involvement of the Cbt transcription factor in Drosophila DC. However its molecular role in the process remained obscure. In this study, we used genomic approaches to identify genes regulated by Cbt as well as its direct targets during late embryogenesis. Our results reveal a complex transcriptional circuit downstream of Cbt and evidence that it is functionally related with the Insulin/insulin-like growth factor signaling pathway. In this context, Cbt may act as a positive regulator of the pathway, leading to the repression of Foxo activity. Our results also suggest that the DC defects observed in cbt embryos could be partially due to Foxo overactivation and that a regulatory feedback loop between Foxo and Cbt may be operating in the DC context.

Transcriptional activity and nuclear localization of Cabut, the Drosophila ortholog of vertebrate TGF-ß-inducible early-response gene (TIEG) proteins.

BACKGROUND: Cabut (Cbt) is a C(2)H(2)-class zinc finger transcription factor involved in embryonic dorsal closure, epithelial regeneration and other developmental processes in Drosophila melanogaster. Cbt orthologs have been identified in other Drosophila species and insects as well as in vertebrates. Indeed, Cbt is the Drosophila ortholog of the group of vertebrate proteins encoded by the TGF-ß-inducible early-response genes (TIEGs), which belong to Sp1-like/Krüppel-like family of transcription factors. Several functional domains involved in transcriptional control and subcellular localization have been identified in the vertebrate TIEGs. However, little is known of whether these domains and functions are also conserved in the Cbt protein. METHODOLOGY/PRINCIPAL FINDINGS: To determine the transcriptional regulatory activity of the Drosophila Cbt protein, we performed Gal4-based luciferase assays in S2 cells and showed that Cbt is a transcriptional repressor and able to regulate its own expression. Truncated forms of Cbt were then generated to identify its functional domains. This analysis revealed a sequence similar to the mSin3A-interacting repressor domain found in vertebrate TIEGs, although located in a different part of the Cbt protein. Using ß-Galactosidase and eGFP fusion proteins, we also showed that Cbt contains the bipartite nuclear localization signal (NLS) previously identified in TIEG proteins, although it is non-functional in insect cells. Instead, a monopartite NLS, located at the amino terminus of the protein and conserved across insects, is functional in Drosophila S2 and Spodoptera exigua Sec301 cells. Last but not least, genetic interaction and immunohistochemical assays suggested that Cbt nuclear import is mediated by Importin-α2. CONCLUSIONS/SIGNIFICANCE: Our results constitute the first characterization of the molecular mechanisms of Cbt-mediated transcriptional control as well as of Cbt nuclear import, and demonstrate the existence of similarities and differences in both aspects of Cbt function between the insect and the vertebrate TIEG proteins.

Planar cell polarity signaling in collective cell movements during morphogenesis and disease.

Collective and directed cell movements are crucial for diverse developmental processes in the animal kingdom, but they are also involved in wound repair and disease. During these processes groups of cells are oriented within the tissue plane, which is referred to as planar cell polarity (PCP). This requires a tight regulation that is in part conducted by the PCP pathway. Although this pathway was initially characterized in flies, subsequent studies in vertebrates revealed a set of conserved core factors but also effector molecules and signal modulators, which build the fundamental PCP machinery. The PCP pathway in Drosophila regulates several developmental processes involving collective cell movements such as border cell migration during oogenesis, ommatidial rotation during eye development, and embryonic dorsal closure. During vertebrate embryogenesis, PCP signaling also controls collective and directed cell movements including convergent extension during gastrulation, neural tube closure, neural crest cell migration, or heart morphogenesis. Similarly, PCP signaling is linked to processes such as wound repair, and cancer invasion and metastasis in adults. As a consequence, disruption of PCP signaling leads to pathological conditions. In this review, we will summarize recent findings about the role of PCP signaling in collective cell movements in flies and vertebrates. In addition, we will focus on how studies in Drosophila have been relevant to our understanding of the PCP molecular machinery and will describe several developmental defects and human disorders in which PCP signaling is compromised. Therefore, new discoveries about the contribution of this pathway to collective cell movements could provide new potential diagnostic and therapeutic targets for these disorders.

Drosophila as a model of wound healing and tissue regeneration in vertebrates.

Understanding the molecular basis of wound healing and regeneration in vertebrates is one of the main challenges in biology and medicine. This understanding will lead to medical advances allowing accelerated tissue repair after wounding, rebuilding new tissues/organs and restoring homeostasis. Drosophila has emerged as a valuable model for studying these processes because the genetic networks and cytoskeletal machinery involved in epithelial movements occurring during embryonic dorsal closure, larval imaginal disc fusion/regeneration, and epithelial repair are similar to those acting during wound healing and regeneration in vertebrates. Recent studies have also focused on the use of Drosophila adult stem cells to maintain tissue homeostasis. Here, we review how Drosophila has contributed to our understanding of these processes, primarily through live-imaging and genetic tools that are impractical in mammals. Furthermore, we highlight future research areas where this insect may provide novel insights and potential therapeutic strategies for wound healing and regeneration.

Mtl interacts with members of Egfr signaling and cell adhesion genes in the Drosophila eye.

Mtl is a member of the Rho family of small GTPases in Drosophila. It was shown that Mtl is involved in planar cell polarity (PCP) establishment, together with other members of the same family like Cdc42, Rac1, Rac2 and RhoA. However, while Rac1, Rac2 and RhoA function downstream of Dsh in Fz/PCP signaling and upstream of a JNK cassette, Mtl and Cdc42 do not. To determine the functional context of Mtl during PCP establishment in the Drosophila eye, we performed a loss-of-function screen to search for dominant modifiers of a sev>Mtl rough eye phenotype. In addition, genetic interaction assays with candidate genes were also carried out. Our results show that Mtl interacts genetically with members and effectors of Egfr signaling, with components and/or regulators of other signal transduction pathways, and with genes involved in cell adhesion and cytoskeleton organization. One of these genes is hibris (hbs), which encodes a member of the immunoglobulin superfamily in Drosophila. Phenotypic analyses and genetic interaction assays suggest that it may have a role during PCP establishment, interacting with both Egfr and Fz/PCP signaling during this process. Taken together, our results indicate that Mtl is functionally related to the Egfr pathway regulating ommatidial rotation during PCP establishment in the eye, being a positive regulator of this pathway. Since Egfr signaling is linked to cytoskeletal and cell junctional elements, it is likely that Mtl may be regulating cytoskeleton dynamics and thus cell adhesion during ommatidial rotation in the context of that pathway.

Expression of Drosophila Cabut during early embryogenesis, dorsal closure and nervous system development.

cabut (cbt) encodes a transcription factor involved in Drosophila dorsal closure (DC), and it is expressed in embryonic epithelial sheets and yolk cell during this process upon activation of the Jun N-terminal kinase (JNK) signaling pathway. Additional studies suggest that cbt may have a role in multiple developmental processes. To analyze Cbt localization through embryogenesis, we generated a Cbt specific antibody that has allowed detecting new Cbt expression patterns. Immunohistochemical analyses on syncytial embryos and S2 cells reveal that Cbt is localized on the surface of mitotic chromosomes at all mitotic phases. During DC, Cbt is expressed in the yolk cell, in epidermal cells and in the hindgut, but also in amnioserosal cells, which also contribute to the process, albeit cbt transcripts were not detected in that tissue. At later embryonic stages, Cbt is expressed in neurons and glial cells in the central nervous system, and is detected in axons of the central and peripheral nervous systems. Most of these expression patterns are recapitulated by GFP reporter gene constructs driven by different cbt genomic regions. Moreover, they have been further validated by immunostainings of embryos from other Drosophila species, thus suggesting that Cbt function during embryogenesis appears to be conserved in evolution.

Dual sex-specific functions of Drosophila Upstream of N-ras in the control of X chromosome dosage compensation.

Dosage compensation in Drosophila melanogaster involves the assembly of the MSL-2-containing dosage compensation complex (DCC) on the single X chromosome of male flies. Translational repression of msl-2 mRNA blocks this process in females. Previous work indicated that the ubiquitous protein Upstream of N-ras (UNR) is a necessary co-factor for msl-2 repression in vitro. Here, we explore the function of UNR in vivo. Hypomorphic Unr mutant flies showed DCC assembly on high-affinity sites in the female X chromosomes, confirming that UNR inhibits dosage compensation in female flies. Unexpectedly, male mutant flies and UNR-depleted SL2 cells showed decreased DCC binding to the X chromosome, suggesting a role for UNR in DCC assembly or targeting. Consistent with this possibility, UNR overexpression resulted in moderate loss of DCC from the male X chromosome and predominant male lethality. Immunoprecipitation experiments revealed that UNR binds to roX1 and roX2, the non-coding RNA components of the DCC, providing possible targets for UNR function in males. These results uncover dual sex-specific functions of UNR in dosage compensation: to repress DCC formation in female flies and to promote DCC assembly on the male X chromosome.

Identification and analysis of cabut orthologs in invertebrates and vertebrates.

Cabut (cbt) is a Drosophila melanogaster gene involved in epidermal dorsal closure (DC). Its expression is dependent on the Jun N-terminal kinase (JNK) cascade, and it functions downstream of Jun regulating dpp expression in the leading edge cells. The Cbt protein contains three C(2)H(2)-type zinc fingers and a serine-rich domain, suggesting that it functions as a transcription factor. We have identified single cbt orthologs in other Drosophila species, as well as in other insects and invertebrate organisms like ascidians and echinoderms, but not in nematodes. Gene structure and protein sequence are highly conserved among Drosophilidae, but are more diverged in the other species of invertebrates analyzed. According to this, we demonstrate that cbt expression is detected in the embryonic lateral epidermis in several Drosophila species, as it occurs in D. melanogaster, thus suggesting that the cbt orthologs may have a conserved role in these species during DC. We have also analyzed the genomes of several vertebrate species, finding that the cbt orthologous genes in these organisms encode proteins that belong to the TIEG family of Sp1-like/Krüppel-like transcription factors. Phylogenetic analysis of the invertebrate and vertebrate proteins identified indicates that they mainly follow the expected phylogeny of the species, and that the cbt gene was duplicated during vertebrate evolution. Because we were not able to identify cbt orthologous genes neither in yeast nor in plants, our results suggest that this gene has been probably conserved throughout metazoans and that it may play a fundamental role in animal biology.